Genetic Identification of Octopodidae Species in Southern California Seafood Markets: Species Diversity and Resource Implications Chase Martin Center for Marine Biodiversity and Conservation Scripps Institution of Oceanography University of California San Diego Abstract Various species of Octopodidae are commonly found in seafood markets throughout Southern California. Most of the octopus available for purchase is imported, with the majority of imports coming from various Asian nations. Despite the diversity of global octopus species, products are most commonly labeled as simply “octopus,” with some distinctions being made in size, e.g., “baby” or “little octopus.” In efforts to characterize species diversity, this study genetically tested 59 octopus samples from a variety of seafood markets in Los Angeles, Orange, and San Diego Counties. Universal 16S rRNA primers (ref) and CO1 primers developed by Folmer et al. (1994) were used for PCR amplification and sequencing of mtDNA. In all, 105 sequences were acquired. Seven species were identified with some confidence. Amphioctopus aegina was the most prevalent species, while two additional species were undetermined. Little available data exists pertaining to octopus fisheries of the countries of production of the samples. Most available information on octopus fisheries pertains to those of Mediterranean and North African nations, and identifies the Octopus vulgaris as the fished species. Characterizing octopus diversity in Southern California seafood markets and assessing labeling and countries of production provides the necessary first step for assessing the possible management implications of these fisheries and seafood supply chain logistics for this group of cephalopods. Introduction Octopuses are exclusively marine cephalopod mollusks that form the order Octopoda. They are found worldwide in various benthic and pelagic habitats, but the highest species diversity occurs in shallow water benthic octopuses that form the family Octopodidae, of which there are most likely over 300 species (Jereb et al., 2014). They are generally fast-growing and short-lived, with the average lifespan lasting one to two years (Jereb et al., 2014). Most species breed only once; males die after mating and the female protects her eggs until their hatching and her subsequent death (Hernández-Garcı́a et al., 2002). Octopus is an exploited marine resource, with global catch reaching upwards of 350,000 tons per year (Jereb et al., 2014). Octopus is highly-valued as a food commodity and bait, though to a lesser degree on the latter (Jereb et al., 2014). Most octopus catch is thought to be Octopus vulgaris from Western Africa and the Mediterranean, and various species from the Western Pacific Ocean (Norman, 2003). Most available information on octopus fisheries refers to those that target O. vulgaris, making it a common label for octopus caught around the world, and it is the most studied of all octopod species (Norman, 2003). Octopus fisheries are diverse, ranging from small-scale artisanal fisheries to large commercial practices. Depending on the fishery, octopus are caught through a variety of methods, including traps, trawls, clay/other types of pots, and simply by hand or by spear (Rathjen, 1991). Despite its lack of any established commercial octopus fishery, the United States has seen increasing market demand for imported octopus (GlobeFish, 2014). Spain is the main provider of US octopus imports, with China and the Philippines occupying top spots as well. In 2010, the US imported 42.7 million dollars worth of octopus, with this number increasing significantly by 2014, reaching 107.8 million dollars. In 2014, over 35 million dollars of octopus imports passed through California alone, with the majority of these products coming into Southern California (NOAA Foreign Trade, 2015). Despite the vast amount of octopus products entering the United States and the evidence for multiple- species fisheries, octopus is commonly only generically labeled without any hint as to what species is being presented. In order to characterize octopus species that make up the market and to better understand fisheries implications, this study aimed to identify octopus samples purchased in seafood markets throughout Southern California. Given the generic labeling of octopus, the study was driven by the question as to how many species exist in the market in Southern California and the trade and fishery implications that might be explored by genetically identifying various octopus samples. Materials and Methods Octopus samples were collected from a variety of seafood markets, restaurants, and retailers in Southern California, specifically Los Angeles, Orange, and San Diego Counties. All samples were raw, and were either frozen or previously frozen and thawed, and sold in whole form, chopped, or mixed with other various seafood products in mixed packages (Fig. 1). Fifty-nine different samples were acquired. Of these fifty-nine, forty- one samples contained multiple individuals. For these purchases, the study selected multiple individuals within the sample to test, depending on the number of specimens available. Multi-individual samples ranged from two to eight specimens within the package. Sample Market Labeling Country of Origin/Production 1 Baby Octopus Vietnam 2 Baby Octopus N/A 3 Baby Octopus India 4 Baby Octopus N/A 5 Octopus NA 6 Octopus Philippines 7 Baby Octopus N/A 8 Octopus N/A 9 Octopus N/A 10 Small Octopus N/A 11 Octopus N/A 12 Small Octopus N/A 13 Octopus New Zealand 14 Baby Octopus USA 15 Baby Octopus Thailand 16 Octopus (as part of seafood mix) China 17 Small Octopus NA 18 Octopus NA 19 Small Octopus China 20 Octopus China 21 Octopus NA 22 Small Octopus China 23 Octopus China 24 Long Arm Octopus China 25 Octopus N/A 26 Octopus Philippines 27 Baby Octopus Vietnam 28 Small Octopus Thailand 29 Small Octopus China 30 Octopus NA 31 Baby Thailand 32 Baby Vietnam 33 Baby Vietnam 34 Little Vietnam 35 Baby India 36 Baby Thailand 37 Little India 38 Octopus China 39 Baby Thailand 40 Baby N/A 41 Baby Thailand 42 Baby Vietnam 43 Baby Thailand 44 Baby N/A 45 Little N/A 46 Octopus N/A 47 Baby N/A 48 Baby N/A 49 Octopus N/A 50 Octopus China 51 Octopus N/A 52 Baby Vietnam 53 Baby Thailand 54 Octopus N/A 55 Octopus N/A 56 Octopus N/A 57 Octopus N/A 58 Octopus N/A 59 Octopus N/A Fig. 1. Table showing sample number, describing how each sample was labeled at the location of purchase, and information on the country of production, if available. DNA was extracted using the QIAGEN DNeasy Blood & Tissue Kit from octopus tissue samples up to 25 mg. Manufacturer protocols for animal tissue were followed for the DNA extraction. The mitochondrial genes for 16S rRNA and CO1 were targeted for amplification by PCR using primers from (Palumbi 1996) and Folmer et al. (1994) respectively. PCR reactions consisted of 12.5 µl of GoTaq Green Master Mix (Promega), 10 pmol of each primer (either for 16S or CO1), and approximately 100 ng of sample DNA in a total volume of 25 µL. The thermal cycler protocol for 16S was as follows: 35 cycles of amplification of the target sequence with an initial denaturation at 95°C for 3 minutes, and post-amplification extension at 72°C for 7 minutes. Each cycle consisted of 30 seconds at 95°C, 1 minute at 50°C, and 1 minute at 72°C. The thermal cycler protocol for CO1 consisted of 5 cycles of amplification followed by 30 additional cycles of amplification under different constraints, with initial denaturation for 3 minutes at 94°C, and post-amplification extension for 5 minutes at 72°C. The first 5 cycles of amplification consisted of 30 seconds at 94°C, 45 seconds at 47°C, and 1 minute at 72°C. The following 30 cycles consisted of 30 seconds at 94°C, 45 seconds at 52°C, and 1 minute at 72°C. Three µL of each PCR product was loaded onto a 2 % agarose gel with gel red to determine the success of the amplification process and then visualized with a UV illuminator. To prepare the PCR product for sequencing, product was cleaned using Sephadex spin column. Resulting DNA was measured using Nanodrop Spectrophotometer ND-1000, and then prepared for sequencing. Sequencing was performed by Retrogen Inc. (San Diego) using the primers described above to sequence the forward strand of each gene. Sequences were trimmed and identity assessed using BLAST at the National Center for Biotechnology Information. Hits that resulted in a 95 percent match or more were taken as positive matches. All sample sequences were edited and aligned in Mesquite (Maddison and Maddison) with top matches from GenBank for the purpose of creating data matrices to determine the degree of relation among the different sequences. Using TCS (Clement) and Popart (Leigh et al.), PAUP* (Swofford) parsimony networks and/or phylogenetic trees (neighbor-joining) were created and visualized. Outgroups were chosen to root the phylogenetic trees by choosing the next most distant BLAST hit. These outgroups were excluded from the network analyses. Fig Tree (Rambaut) was used to examine phylogenetic trees and Popart networks were used to assess the degree of relation between sequences and their countries of production. Results Sample Top GenBank Match 1B A. marginatus 95% 22A O. minor 99% 2 A. aegina 99% 22B O. minor 97% O. sp. BOLD:ABA1887 23A O. minor 98% 3A 99% 23B O. minor 99% 4A A. aegina 99% 24 O. minor 97% 4B A. aegina 100% 25A O. minor 99% 5 O. cyanea 99% 25B O. minor 98% 6 O. cyanea 99% 26 O. cyanea 99% 7B O. cyanea 98% O. sp. BOLD:ABA1887 8 O. minor 100% 27A 98% 9A O. minor 99% 27B A. marginatus 94% 9B O. minor 99% O.